JP5830249B2 - Method for determining sensitivity of cancer cells to anthracycline anticancer drugs and computer program - Google Patents

Description

  The present invention relates to a method and a computer program for determining the sensitivity of cancer cells to an anthracycline anticancer agent based on the expression level of GSTπ.

  Chemotherapy is one of the common treatment methods for cancer, and various anticancer agents are administered. However, effective anticancer agents differ depending on the type of cancer and individual patient, and anticancer agents often have overlapping therapeutic and harmful areas. Therefore, when an ineffective anticancer drug is administered, not only the possibility of recurrence of the cancer increases, but the physical strength of the patient decreases due to side effects of the anticancer drug, and even if another effective anticancer drug is administered next, sufficient efficacy is exhibited. There is a fear that it cannot be done. For this reason, in order to obtain efficacy while ensuring safety, development of a system that predicts drug reactivity that accurately identifies an effective anticancer agent before administration is eagerly desired.

  Conventionally, as a sensitivity test for anticancer drugs against cancer, a method has been adopted in which various anticancer drugs are brought into contact with cancer cells isolated from patients, and anticancer drugs that are considered to be effective for cancer cells are used as indicators of cancer cell growth inhibition. It has been. However, in such a trial and error method, the test results do not sufficiently reflect the clinical therapeutic effect, and a large amount of cancer cells are required for anticancer drug treatment. Therefore, there have been problems of recurrence due to administration of an anticancer agent having a low therapeutic effect, and a burden on patients due to collection of a large amount of cancer cells.

  Anthracycline anticancer agents are anticancer agents that induce apoptosis by inhibiting recombination of topoisomerase II DNA. Anthracycline anticancer agents are highly evaluated clinically and are widely used in chemotherapy such as breast cancer. However, like other anticancer agents, it may cause serious side effects such as cardiotoxicity and leukopenia. Therefore, the establishment of a method for determining the sensitivity of cancer cells to anthracycline anticancer agents is very important.

  Glutathione-S-transferase π (GSTπ) is a protein having a molecular weight of about 22.5 kDa encoded by the GSTP1 gene among enzymes belonging to the human glutathione-S-transferase (GST) family. GSTπ is involved in attenuating the toxicity of anticancer agents by catalyzing the formation of conjugates between reduced glutathione (GSH) and anticancer agents. Therefore, it is generally known that cancer cells in which GSTπ is highly expressed have higher resistance to anticancer agents than cancer cells in which GSTπ is lowly expressed. For example, Non-Patent Document 1 reports that cancer cells in which GSTπ is generally highly expressed are more resistant to anthracycline anticancer agents than cancer cells in which GSTπ is lowly expressed.

Andrew H., et al. ROLE OF GLUTATHIONE S-TRANSFERASE P1, P-GLYCOPROTEIN AND MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 1 IN ACQUIRED DOXORUBICIN RESISTANCE.Int.J.Cancer: 92,777-783 (2001)

An object of the present invention is to provide a method for determining the sensitivity of cancer cells of breast cancer to an anthracycline anticancer agent, and a computer program for causing a computer to execute the method.

As described above, conventionally, cancer cells in which GSTπ is highly expressed were considered to have increased resistance to anthracycline anticancer agents. However, the present inventors have surprisingly found that the cancer cells of breast cancer GSTπ is highly expressed is found that higher susceptibility to breast cancer cells of an anthracycline anticancer agent, and completed the present invention Arrived.

That is, the present invention compares the step of measuring the expression level of GSTπ contained in a biological sample containing cancer cells of breast cancer with the first threshold value of the expression level of GSTπ obtained in the measurement step. when the expression level is greater than or equal to the first threshold value includes the step of determining a susceptibility to breast cancer cells anthracycline anticancer agent is highly contained in a biological sample, a breast cancer that is contained in a biological sample cancer cells A method for determining sensitivity to an anthracycline anticancer agent is provided.
Further, the present invention provides a computer, an acquisition means for acquiring an expression level of GSTπ contained in a biological sample containing cancer cells of breast cancer, a comparison means for comparing the expression level of GSTπ with a first threshold, and GSTπ when the expression level is greater than or equal to the first threshold, and sensitive and determining means to cancer cells of anthracycline anticancer agent for breast cancer contained in the biological sample, output means for outputting a determination result function as, A computer program is provided.

According to the present invention, it is possible to provide an objective and accurate method for determining the sensitivity of cancer cells of breast cancer to an anthracycline anticancer agent without imposing a large burden on the patient. In addition, according to the present invention, it is possible to provide a computer program for causing a computer to execute the above-described sensitivity determination method.

Further, according to the method and computer program of the present invention, based on the expression amount of GSTπ in breast cancer cells taken from breast cancer patients, breast cancer to cancer patients, it can determine anthracycline anticancer agent sensitivity. Therefore, it is possible not only to administer an effective anthracycline anticancer agent to a patient but also to avoid unnecessary administration of an anthracycline anticancer agent. As a result, according to the present invention, it is possible to reduce the burden due to the side effects of the patient.

1 shows an embodiment of a computer that determines the sensitivity of cancer cells contained in a biological sample to an anthracycline anticancer agent. The determination flow in the computer which determines that the sensitivity to the anthracycline anticancer agent using the expression level of GSTπ is high is shown. The determination flow in the computer which determines with the sensitivity to an anthracycline anticancer agent using the expression level of GST (pi) and a CDK1 specific activity value / CDK2 specific activity value is high is shown. The determination flow in the computer which determines with the sensitivity to an anthracycline anticancer agent using the expression level of GST (pi) and a CDK1 specific activity value / CDK2 specific activity value is high or low is shown. In Example 1, it is a figure which shows the result of IC50 and the expression level of GST (pi) in 29 types of breast cancer cell lines. In Example 2, it is a figure which shows the expression level of GST (pi) in a non-relapsed sample and a recurrent sample among nine clinical samples. In Example 3, it is a figure which shows the result of IC50 in 27 types of breast cancer cell lines, the expression level of GST (pi), and the specific activity value of CDK2 / specific activity value of CDK1.

  In the present embodiment, the cancer cells are not particularly limited. Examples thereof include cancer cells such as lung cancer, stomach cancer, colon cancer, ovarian cancer, brain tumor, breast cancer, prostate cancer, skin cancer and leukemia cells. In this embodiment, the cancer cell is preferably a cancer cell of breast cancer.

  In the present embodiment, the cancer patient is not particularly limited. Examples thereof include cancer patients such as lung cancer, stomach cancer, colon cancer, ovarian cancer, brain tumor, breast cancer, prostate cancer, skin cancer, and leukemia cells. In this embodiment, the cancer patient is preferably a cancer patient with breast cancer.

  In the present embodiment, the biological sample is not particularly limited as long as it is a sample including a plurality of cells collected from animals such as humans. For example, blood, serum, lymph, urine, nipple discharge, and cells or tissues collected from subjects by surgery or biopsy. In addition, a sample obtained by culturing cells or tissue collected from a subject can also be used as a biological sample.

  In the present embodiment, the anthracycline anticancer agent is not particularly limited as long as it is an anthracycline compound having an anticancer activity. For example, daunorubicin, doxorubicin, pirarubicin, aclarubicin, epirubicin, oxaunomycin, idarubicin. In the present embodiment, the anthracycline anticancer agent is preferably doxorubicin.

  The present embodiment includes a step of measuring the expression level of GSTπ contained in a biological sample containing cancer cells (hereinafter sometimes referred to as “measurement step”). The measurement step may be performed using a known measurement method capable of measuring the expression level of GSTπ protein or mRNA, and is not particularly limited. Known measurement methods include, for example, SDS polyacrylamide electrophoresis, two-dimensional electrophoresis, protein chip analysis, enzyme-linked immunoassay (ELISA), immunofluorescence, western blotting, dot blotting, Examples thereof include an immunoprecipitation method, RT-PCR method, Northern blotting method, NASBA method, and DNA chip method. As a measuring method in the present embodiment, a measuring method for measuring the expression level of GSTπ protein is preferable.

  This embodiment is a step of determining that cancer cells contained in a biological sample are highly sensitive to an anthracycline anticancer agent when the expression level of GSTπ obtained in the measurement step is large (hereinafter referred to as “determination step”). Is included).

  In the present embodiment, the magnitude of the expression level of GSTπ can be determined by visually or numerically expressing the expression level of GSTπ according to the measurement method used in the measurement process. For example, in the measurement process, when the expression level of GSTπ protein is measured by Western blotting, the GSTπ protein band appearing on the membrane is visually compared with the control band, and the GSTπ expression level is large or small. Can be determined. In addition, the GSTπ protein band that appears on the membrane is incorporated into an image scanner, and the signal intensity is analyzed to quantify the expression level of GSTπ. Alternatively, in the measurement step, when the expression level of GSTπ protein is measured using a filter plate, the fluorescence of the GSTπ protein that appears on the plate and the control fluorescence are visually compared to determine the expression level of GSTπ. You can determine the size. In addition, the GSTπ fluorescence appearing on the above-described plate is taken into an image scanner, and the fluorescence intensity is analyzed to quantify the expression level of GSTπ. The magnitude of the expression level of GSTπ can also be determined by comparing the digitized expression level of GSTπ with a predetermined threshold value (first threshold value). In the determination step in the present embodiment, it is preferable from the viewpoint of objectivity to compare the digitized expression level of GSTπ with the first threshold value and determine the magnitude of the expression level of GSTπ.

  Here, the first threshold value is a criterion for determining the expression level of GSTπ when determining the sensitivity of cancer cells contained in a biological sample to an anthracycline anticancer agent. This first threshold value can be appropriately set empirically according to the measurement method used in the measurement process. For example, the expression level of GSTπ is measured in a plurality of biological samples containing cancer cells highly sensitive to an anthracycline anticancer agent. Similarly, the expression level of GSTπ in a plurality of biological samples containing cancer cells that are low in sensitivity to anthracycline anticancer agents is measured. A GSTπ that can distinguish between a biological sample containing cancer cells highly sensitive to an anthracycline anticancer agent and a biological sample containing cancer cells less sensitive to an anthracycline anticancer agent from the expression levels of the plurality of GSTπ obtained. Can be set as the first threshold value.

  In the present embodiment, the measurement step further includes a specific activity value of cyclin-dependent kinase 1 (CDK1) and a specific activity value of cyclin-dependent kinase 2 (CDK2) contained in a biological sample containing cancer cells. Can be measured. In addition, in the determination step, when the expression level of GSTπ obtained in the measurement step is large, or the specific activity value of CDK2 obtained in the measurement step / the specific activity value of CDK1 obtained in the measurement step (hereinafter, the CDK2 specific activity value) / CDK1 specific activity value) and the second threshold value, and when the CDK2 specific activity value / CDK1 specific activity value is larger than the second threshold value, the cancer cell contained in the biological sample It can be determined that the sensitivity to anthracycline anticancer agents is high. Thus, by using the value of CDK2 specific activity value / CDK1 specific activity value in addition to the expression level of GSTπ, the sensitivity of cancer cells to an anthracycline anticancer agent can be determined more accurately.

  Here, the specific activity value of CDK1 corresponds to the ratio of CDK1 showing activity in CDK1 present in cells. Specifically, the specific activity value of CDK1 can be calculated from the activity value of CDK1 / the expression level of CDK1. The specific activity value of CDK2 corresponds to the proportion of CDK2 exhibiting activity in CDK2 present in cells. Specifically, the specific activity value of CDK2 can be calculated from the activity value of CDK2 / the expression level of CDK2.

  The activity value of CDK1 and the activity value of CDK2 are, for example, the level of kinase activity (unit is represented by U (unit)) calculated from the amount of substrate phosphorylated by CDK. An example of a substrate that phosphorylates CDK1 and CDK2 is histone H1.

The activity value of CDK1 and the activity value of CDK2 can be measured by a known CDK activity measurement method. Known methods for measuring CDK activity include, for example, a method using a label with a radioactive substance and a method without using a label with a radioactive substance. As a method of using a labeling with radioactive materials, for example, a sample was prepared from a biological sample containing cancer cells, using the relevant specimen and the ³² P-labeled ^{ATP ([γ- 32 P] ATP} ), the matrix protein ³² Examples include a method in which P is incorporated, the amount of ³² P-labeled phosphorylated substrate is measured, and the activity value of CDK1 or the activity value of CDK2 is quantified based on a calibration curve prepared with a standard product. Moreover, as a method which does not use the label | marker by a radioactive substance, the method as described in US Publication 2002/164673 is mentioned, for example. In the method described in US Publication No. 2002/164673, a sample is prepared from a biological sample containing cancer cells, adenosine 5′-O- (3-thiotriphosphate) (ATPγS) is reacted with a substrate protein, The substrate protein is labeled by introducing a monothiophosphate group into the serine residue or threonine residue of the substrate protein, and binding a fluorescent labeling substance or labeling enzyme to the sulfur atom of the introduced monothiophosphate group, and labeled thiophosphate In this method, the amount of labeling based on the group (fluorescence amount when a fluorescent labeling substance is used) is measured and quantified based on a calibration curve prepared with a standard product.

  A sample used for activity measurement is prepared by specifically collecting CDK1 or CDK2 from a biological sample containing cancer cells to be measured. In this case, the sample is prepared using an anti-CDK antibody specific for either CDK1 or CKD2. In either case, CDK1 other than activated CDK1 or CKD2 other than activated CDK2 will be included. For example, in the case of CKD1, a complex in which a CDK1 inhibitor is bound to a cyclin / CDK1 complex is also included. When an anti-CDK1 antibody is used, a single CDK1, a complex of CDK1 and cyclin and / or a CDK1 inhibitor, a complex of CDK1 and other compounds, and the like are included. Therefore, the CDK1 activity value is measured as a unit (U) of a phosphorylated substrate in a state where CDK1 active type, CDK1 inactive type, and various competitive reactions coexist. In the case of CDK2, measurement is performed under the same conditions as in the case of CKD1 described above.

  The expression level of CDK1 or the expression level of CDK2 is the amount of CDK1 or CDK2 (unit corresponding to the number of molecules) contained in the solubilized solution of the biological sample containing the cancer cells to be measured. It can measure by a conventionally well-known method of measuring. For example, an ELISA method, a Western blot method, or the like may be used, and the measurement may be performed by the method disclosed in JP-A-2003-130971. CDK1 and CDK2 may be captured using specific antibodies. For example, in the case of CDK1, by using an anti-CDK1 antibody, all of CDK1 existing in the cell (including CDK1 carrier, complex of CDK1 and cyclin and / or CDK1 inhibitor, complex of CDK1 and other compounds) ) Can be supplemented. In the case of CDK2, the expression level of CDK2 can be measured by the same method as in the case of CKD1 described above.

  Here, the second threshold is a criterion for determining the CDK2 specific activity value / CDK1 specific activity value when determining the sensitivity of cancer cells contained in a biological sample to an anthracycline anticancer agent. This threshold value can be appropriately set empirically according to the measurement method used in the measurement process. For example, the CDK2 specific activity value / CDK1 specific activity value of a plurality of biological samples containing cancer cells highly sensitive to an anthracycline anticancer agent is measured. Similarly, the CDK2 specific activity value / CDK1 specific activity value of a plurality of biological samples containing cancer cells with low sensitivity to an anthracycline anticancer agent is measured. A biological sample containing cancer cells highly sensitive to anthracycline anticancer agents and a biological sample containing cancer cells less sensitive to anthracycline anticancer agents from the obtained multiple CDK2 specific activity values / CDK1 specific activity values Can be set as a threshold value that can be distinguished from the CDK2 specific activity value / CDK1 specific activity value.

  In the present embodiment, the determination step further compares the CDK2 specific activity value / CDK1 specific activity value with the second threshold value, and the expression level of GSTπ obtained in the measurement step is small. When the value of the CDK1 specific activity value is smaller than the threshold value, it can be determined that the sensitivity of the cancer cells contained in the biological sample to the anthracycline anticancer agent is low. Thus, by using the expression level of GSTπ and the value of CDK2 specific activity value / CDK1 specific activity value, it can also be determined that the sensitivity of cancer cells to anthracycline anticancer agents is low. As a result, unnecessary administration of an anthracycline anticancer agent to the patient can be avoided.

  In the present embodiment, the computer program can cause a computer to determine the sensitivity of cancer cells contained in the biological sample to an anthracycline anticancer agent. An example of the specific computer is shown in FIG.

  The computer 100 mainly includes a main body 110, a display unit 120, and an input device 130. In the main body 110, a CPU 110a, a ROM 110b, a RAM 110c, a hard disk 110d, a reading device 110e, an input / output interface 110f, and an image output interface 110h are connected to each other via a bus 110i so that data communication is possible.

The CPU 110a can execute computer programs stored in the ROM 110b and computer programs loaded in the RAM 110c.
The ROM 110b is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and is recorded with a computer program executed by the CPU 110a and data used therefor.
The RAM 110c is configured by SRAM, DRAM, or the like. The RAM 110c is used to read out computer programs recorded in the ROM 110b and the hard disk 110d. Further, when these computer programs are executed, they are used as a work area of the CPU 110a.

  The hard disk 110d is installed with various computer programs to be executed by the CPU 110a such as an operating system and application system programs, and data used for executing the computer programs. An application program 140a described later is also installed in the hard disk 110d.

  The reading device 110e is configured by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read a computer program or data recorded on the portable storage medium 140. Further, the portable storage medium 140 stores an application program 140a related to determination in the computer, and the CPU 110a reads the application program 140a from the portable storage medium 140 and installs the application program 140a in the hard disk 110d. Is possible.

  In addition, an operating system that provides a graphical user interface environment such as Windows (registered trademark) manufactured and sold by US Microsoft Corporation is installed in the hard disk 110d. In the following description, it is assumed that the application program 140a related to the determination described above operates on the operating system.

  The input / output interface 110f includes, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog interface including a D / A converter, an A / D converter, and the like. ing. An input device 130 including a keyboard and a mouse is connected to the input / output interface 110f, and the user can input data to the computer main body 110 by using the input device 130.

  Further, the measuring device 200 is connected to the input / output interface 110f. Thereby, the computer main body 110 can acquire the expression level of GSTπ from the measurement apparatus 200 via the input / output interface 110f.

  The image output interface 110h is connected to a display unit 120 configured by an LCD, a CRT, or the like, and outputs a video signal corresponding to image data given from the CPU 110a to the display unit 120. The display unit 120 outputs image data according to the input video signal. Further, the display unit 120 outputs a determination result given from a CPU 110a described later.

  FIG. 2 is a flowchart showing the operation of the application program 140a for executing the above-described determination based on the expression level of GSTπ of cancer cells contained in the biological sample. First, information regarding the expression level of GSTπ of cancer cells contained in a biological sample acquired by the measuring apparatus 200 is input to the computer main body 110 via the input / output interface 110f. The CPU 110a calculates the GSTπ expression level based on the information related to the GSTπ expression level, and stores it in the RAM 110c (step S1). Here, as information regarding the expression level of GSTπ of cancer cells contained in a biological sample, the fluorescence intensity measured by a fluorescence intensity measuring apparatus (Plate Reader (Infinite F200, manufactured by Tecan)) in Example 1 described later is a computer. Input to the main body 110.

  The CPU 110a calls a threshold value stored in advance in the memory 110d as data of the application program 140a, and compares this threshold value with the expression level of GSTπ (step S2). Here, the threshold value is the threshold value 3 of the GSTπ expression level in Example 1 described later.

  Next, the CPU 110a determines whether or not the expression level of GSTπ is greater than or equal to a threshold value (step S3).

  Next, when the expression level of GSTπ is equal to or greater than the threshold value, the CPU 110a determines that the sensitivity of the cancer cells contained in the biological sample to the anthracycline anticancer agent is “high” (step S4). Conversely, if the expression level of GSTπ is less than the threshold, the CPU 110a sets the determination of the sensitivity of the cancer cells contained in the biological sample to the anthracycline anticancer agent as “pending” (step S5). Here, among 29 types of breast cancer cell lines in Example 1 described later, 9 types of breast cancer cell lines were determined to be “high” in sensitivity to anthracycline anticancer agents, and 20 types of breast cancer cell lines were “reserved”. It was determined.

  The CPU 110a stores the determination result in the RAM 110c and outputs it to the display unit 120 via the image output interface 110h (step S6).

  In the present embodiment, the expression level of GSTπ is acquired from the measurement apparatus 200 via the input / output interface 110f. However, the present invention is not limited to this. For example, the expression level of GSTπ is input by the operator. Thus, it may be acquired from the input device 130.

  In addition to the expression level of GSTπ, the computer program in the present embodiment further uses the value of the specific activity value of CDK2 / specific activity value of CDK1 (CDK2 specific activity value / CDK1 specific activity value) to determine the ansla of cancer cells. The computer can determine the sensitivity to the cyclin anticancer agent.

  FIG. 3 is a flowchart showing the operation of the application program 140a that determines that a cancer cell is highly sensitive to an anthracycline anticancer agent based on the expression level of GSTπ and the CDK1 specific activity value / CDK2 specific activity value. First, information regarding the expression level of GSTπ, the activity value of CDK1, the expression value of CDK1, the activity value of CDK2, and the expression level of CDK2 of cancer cells contained in the biological sample acquired by the measuring apparatus 200 is input to the input / output interface 110f. To the computer main body 110. The CPU 110a determines the expression level of GSTπ, the activity value of CDK1, the activity value of CDK1, the activity value of CDK1, the activity value of CDK1, the activity value of CDK1, The value and the expression level of CDK2 are calculated and stored in the RAM 110c (step S7). Here, as information on the GSTπ expression level, CDK1 activity value, CDK1 expression level, CDK2 activity level, and CDK2 expression level of cancer cells contained in the biological sample, a fluorescence intensity measuring device (Example 3 described later) ( The fluorescence intensity measured by the plate reader (Infinite F200, manufactured by Tecan) is input to the computer main body 110.

  The CPU 110a calculates the value of CDK2 specific activity value / CDK1 specific activity value from the CDK1 activity value, CDK1 expression level, CDK2 activity level, and CDK2 expression level stored in the RAM 110c (step S8).

  The CPU 110a calls the first threshold value and the second threshold value stored in advance in the memory 110d as the data of the application program 140a, and the first threshold value and the expression level of GSTπ, and the second threshold value and the CDK2 specific activity value A comparison with the value of the / CDK1 specific activity value is performed. (Step S9). Here, the first threshold value is the threshold value 3 of the GSTπ expression level in Example 3 described later. The second threshold value is the threshold value 7.41 of the value of CDK2 specific activity value / CDK1 specific activity value in Example 3 described later.

  Next, the CPU 110a determines whether or not the expression level of GSTπ is equal to or greater than the first threshold value, or the value of the CDK2 specific activity value / CDK1 specific activity value is equal to or greater than the second threshold value (step S10).

  Next, when the expression level of GSTπ is equal to or higher than the first threshold value, or when the value of CDK2 specific activity value / CDK1 specific activity value is equal to or higher than the second threshold value, the CPU 110a It is determined that the sensitivity to an anthracycline anticancer agent is “high” (step S11). Conversely, when the expression level of GSTπ is less than the first threshold value, or when the value of CDK2 specific activity value / CDK1 specific activity value is less than the second threshold value, CPU 110a determines that the cancer cell contained in the biological sample The determination of the sensitivity to an anthracycline anticancer agent is “pending” (step S12). Here, of the 27 breast cancer cell lines in Example 3 described later, 16 breast cancer cell lines were determined to be “high” to anthracycline anticancer agents, and 11 breast cancer cell lines were “reserved”. It was determined.

  Then, the CPU 110a stores the determination result in the RAM 110c and outputs it to the display unit 120 via the image output interface 110h (step S13).

  Furthermore, in addition to the expression level of GSTπ, the computer program according to the present embodiment uses a value of CDK2 specific activity value / CDK1 specific activity value to further select cancer cells that are “low” to anthracycline anticancer agents, It can also be determined.

  FIG. 4 is a flowchart showing the operation of the application program 140a that determines that the sensitivity of the cancer cell to the anthracycline anticancer agent is high or low based on the expression level of GSTπ and the CDK1 specific activity value / CDK2 specific activity value. First, information regarding the expression level of GSTπ, the activity value of CDK1, the expression value of CDK1, the activity value of CDK2, and the expression level of CDK2 of cancer cells contained in the biological sample acquired by the measuring apparatus 200 is input to the input / output interface 110f. To the computer main body 110. The CPU 110a determines the GSTπ expression level, the CDK1 activity level, the CDK1 expression level, the CDK2 activity level, and the CDK2 expression level based on the GSTπ expression level, the CDK1 activity level, the CDK1 expression level, and the CDK2 activity level. And the expression level of CDK2 is calculated and stored in the RAM 110c (step S14). Here, as information on the GSTπ expression level, CDK1 activity value, CDK1 expression level, CDK2 activity level, and CDK2 expression level of cancer cells contained in the biological sample, a fluorescence intensity measuring device (Example 3 described later) ( The fluorescence intensity measured by the plate reader (Infinite F200, manufactured by Tecan) is input to the computer main body 110.

  The CPU 110a calculates the value of CDK2 specific activity value / CDK1 specific activity value from the CDK1 activity value, CDK1 expression level, CDK2 activity level, and CDK2 expression level stored in the RAM 110c (step S15).

  The CPU 110a calls the first threshold value and the second threshold value stored in advance in the memory 110d as the data of the application program 140a, and the first threshold value and the expression level of GSTπ, and the second threshold value and the CDK2 specific activity value A comparison with the value of the / CDK1 specific activity value is performed. (Step S16). Here, the first threshold value is the threshold value 3 of the GSTπ expression level in Example 3 described later. The second threshold value is the threshold value 7.41 of the value of CDK2 specific activity value / CDK1 specific activity value in Example 3 described later.

  Next, the CPU 110a determines whether or not the expression level of GSTπ is less than the first threshold value and the value of the CDK2 specific activity value / CDK1 specific activity value is less than the second threshold value (step S17).

  When the expression level of GSTπ is less than the first threshold value and the value of the CDK2 specific activity value / CDK1 specific activity value is less than the second threshold value, the CPU 110a becomes an anthracycline anticancer agent for cancer cells contained in the biological sample. Is determined to be “low” (step S18). Conversely, when the expression level of GSTπ is greater than or equal to the first threshold, or when the value of the CDK2 specific activity value / CDK1 specific activity value is greater than or equal to the second threshold, the CPU 110a determines whether the cancer cell anthrax contained in the biological sample. It is determined that the sensitivity to the cyclin anticancer agent is “high” (step S19). Here, of the 27 types of breast cancer cell lines in Example 3 described later, 7 types of breast cancer cell lines were determined to be “low” to anthracycline anticancer agents, and 20 types of breast cancer cell lines were anthracycline anticancer agents. Sensitivity to was determined to be “high”.

  Then, the CPU 110a stores one of the above determination results in the RAM 110c and outputs it to the display unit 120 via the image output interface 110h (step S20).

  In this embodiment, the expression level of GSTπ, the CDK1 activity value, the CDK1 expression level, the CDK2 activity value, and the CDK2 expression level were acquired from the measurement apparatus 200 via the input / output interface 110f. For example, the expression level of GSTπ may be acquired from the input device 130 by an input by the operator.

  In the present embodiment, the expression level of GSTπ is acquired from the measurement apparatus 200 via the input / output interface 110f. However, the present invention is not limited to this. For example, the expression level of GSTπ is input by the operator. Thus, it may be acquired from the input device 130.

(Reference Example 1)
<Culture of breast cancer cell lines>
Human breast cancer cell lines AU565 cell (ATCC CRL-2351), BT-20 cell (ATCC HTB-19), BT-474 cell (ATCC HTB-20), BT-549 cell (ATCC HTB-122), HCC1569 cell (ATCC CRL-2330), HCC1937 cells (ATCC CRL-2336), Hs578T cells (ATCC HTB-126), SK-BR-3 cells (ATCC HTB-30), ZR-75-1 cells (ATCC CRL-1500) , ZR-75-30 cells (ATCC CRL-1504), HCC1419 cells (ATCC CRL-2326), HCC202 cells (ATCC CRL-2316), BT-483 cells (ATCC HTB-121), CAMA-1 cells (ATCC HTB) -21), HC 1954 cells (ATCC CRL-2338), MCF7 cells (ATCC HTB-22), T-47D cells (ATCC HTB-133), HCC1500 cells (ATCC CRL-2329), HCC1428 cells (ATCC CRL-2327), HCC1806 cells ( ATCC CRL-2335), MDA-MB-415 cells (ATCC HTB-128), MDA-MB-231 cells (ATCC HTB-26), MDA-MB-361 cells (ATCC HTB-27), MDA-MB-435S Cells (ATCC HTB-129), MDA-MB-453 cells (ATCC HTB-131), UACC-812 cells (ATCC CRL-1897), MDA-MB-157 cells (ATCC HTB-24), MDA-MB-436 Cell (ATC HTB-130), UACC-893 cells (ATCC CRL-1902), were purchased from American Type Culture Collection (ATCC), were cultured according to the culture conditions of the ATCC recommended.

  Table 1 shows the basic culture conditions recommended by ATCC for each cell line, the FBS (fetal calf serum) percent concentration in the basic culture medium, the additives to the culture medium, and the culture environment.

(Reference Example 2)
<Preparation of measurement sample>
Each of the 29 types of breast cancer cell lines shown in Reference Example 1 was seeded in a medium in a 15 cm dish under the culture conditions of Reference Example 1, and cultured to 70-80% confluent. After culturing, the medium was removed from the 15 cm dish, 10 mL of phosphate buffered saline was placed in the 15 cm dish, and the cells in the 15 cm dish were washed with the phosphate buffered saline. Next, 3 mL of trypsin-EDTA solution (0.05% trypsin, 0.53 mM EDTA) was added to the cells in the 15 cm dish, incubated at 37 ° C. for 5-10 minutes, and the cells were detached from the flask. To this flask, 10 mL or more of medium was added to stop the reaction with trypsin, and the cells were collected.
The collected cells were each transferred to a microtube. Next, a solubilizing reagent (50 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 50 mM NaF, 1 mM Na ₃ VO ₄ , 0.1% NP40, 0.2% protease inhibitor cocktail (sigma)) ) Was dispensed at 150 to 250 μL so that the protein concentration in the solution was 2 ug / uL. Next, the microtube was placed in a homogenizer and then homogenized, and centrifuged at 15000 rpm for 5 minutes at 4 ° C. with a micro high-speed centrifuge CF15RX (manufactured by Hitachi). Next, the supernatant of the microtube was transferred to a 1.5 mL microtube placed on ice to prepare a measurement sample.

  (Example 1)

<Determination of susceptibility of cancer cells to anthracycline anticancer agents by GSTπ expression level>
The determination of the sensitivity of cancer cells to anthracycline anticancer agents by the GSTπ expression level of the cancer cells contained in the biological sample was examined as follows.

<Measurement of IC50>
Each of the 29 types of breast cancer cell lines shown in Reference Example 1 is 5000 cells / 100 uL / well on two microplates in a medium containing doxorubicin at concentrations of 0 nM, 250 nM, 500 nM, 750 nM, 1000 nM, and 2000 nM. And seeded for 24 hours.
Next, a medium containing each concentration of doxorubicin was prepared, and the medium was replaced. One of the microplates was subjected to cell proliferation measurement using Celltiter Glo Luminescent Cell Viavility Assay (manufactured by Promega: Cat # G7571) immediately after medium replacement. After the medium was replaced, the remaining one was cultured for 72 hours under the culture conditions shown in Reference Example 1, and then cell proliferation was measured using Celltiter Glo Luminescent Cell Viavility Assay (Promega: Cat # G7571). .

  Based on the measured number of viable cells, the number of viable cells in the medium not treated with doxorubicin / the number of viable cells in the medium obtained by treatment with doxorubicin × 100 = 50 concentration of doxorubicin (IC50) was determined. .

  Here, the median value of IC50, 392 nM, was set as a threshold, a breast cancer cell line showing a threshold value or more was set as an insensitive strain, and a breast cancer cell line showing a threshold value less than the threshold was set as a sensitive strain.

<Measurement of GSTπ expression level>
The reagent to be used was prepared as follows.
The reagents used for the measurement of the expression level of GSTπ are shown below.
(1) Blocking reagent
Block Ace (powder) (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) was dissolved in ultrapure water to a final concentration of 4% to prepare a blocking reagent.

(2) Primary antibody reagent
A GSTπ antibody (BD Transduction) was dissolved in a blocking solution diluted 10-fold so as to be 5 ug / mL to prepare a primary antibody reagent.

(3) Tris buffered saline (TBS)
TBS was prepared by diluting 10 × TBS solution (2-Amino-2-hydroxymethyl-1,3-propanediol: 30.28 g, NaCl: 87.7 g, 6N HCl: 110 g / L) 10 times.

(4) Secondary antibody reagent
Bovine serum albumin BSA was dissolved in TBS in ultrapure water to a final concentration of 1% to prepare TBS (1% BSA). Dissolve Goat Anti-Mouse IgG (H + L) -BIOT Human / Mouse (Southern Biotech) at 30 ug / mL in the resulting TBS (1% BSA) to prepare the secondary antibody reagent. did.

(4) Fluorescent labeling reagent Fluorescein StreptAvidin (manufactured by Vector) was dissolved in TBS (1% BSA) described above to a concentration of 10 μg / mL to prepare a fluorescent labeling reagent.

  The expression level of GSTπ was measured as follows. 10 μg / well of each measurement sample of 29 types of breast cancer cell lines shown in Reference Example 1 prepared as in Reference Example 2 was prepared on a filter plate (Multi Screen HTS PSQ Plates), and water was removed by aspiration. Thereafter, 100 μL / well of blocking agent reagent was dispensed and aspirated.

  After aspiration, the primary antibody reagent was dispensed at 50 μL / well and immediately aspirated, then again dispensed at 50 uL / well, allowed to stand for 2 hours, and then aspirated to remove the primary antibody. Next, TBS 300 μL / well was dispensed and suctioned 4 times. After washing, the secondary antibody reagent was dispensed at 50 μL / well and immediately aspirated, then again dispensed at 50 uL / well, allowed to stand for 1 hour, and then aspirated to remove the secondary antibody. Next, TBS 300 μL / well was dispensed and suction was washed twice. After washing, the standard fluorescent labeling reagent was dispensed at 100 μL / well and aspirated immediately after. Next, TBS 300 μL / well was dispensed and suctioned four times, and then the filter plate was dried.

  After drying, the fluorescence intensity was measured with InfiniteF200 (Tecan). Here, the expression level of GSTπ of each measurement sample is 0 ng / 100 uL / well, 10 ng / 100 uL / well, 30 ng / 100 uL / well, 50 ng / 100 GSTP1 recombinant Protein (Abnova Cat No.H00002950-P01) as a calibrator. It was calculated using a calibration curve prepared by placing on a filter plate so as to have each concentration of 100 uL / well and measuring fluorescence intensity with InfiniteF200 (manufactured by Tecan).

  Here, the threshold value for the expression level of GSTπ was set to 3, a breast cancer cell line exhibiting a threshold value or higher was designated as a GSTπ high expression strain, and a breast cancer cell line exhibiting less than the threshold was designated as a GST low expression strain.

  The results of this example are shown in FIG.

  From the results shown in FIG. 5, among 29 types of breast cancer cell lines, the GSTπ high-expression cell lines tended to be highly sensitive to doxorubicin. As a result, it was shown that when the expression level of GSTπ in cancer cells is large, it can be determined that the sensitivity of cancer cells to anthracycline anticancer agents is high.

  (Example 2)

<GSTπ expression level in recurrent specimens and non-recurrent specimens>
Next, from the GSTπ expression level of 9 breast cancer patients before receiving chemotherapy containing an anthracycline anticancer agent, the GSTπ expression level in relapsed and non-recurrent specimens receiving chemotherapy containing an anthracycline anticancer agent after surgery. The trend was examined.

The nine specimens were tumor tissues including breast cancer that had been surgically excised, and were stored frozen at −80 ° C. until measurement. Of the 9 samples, 5 were recurrent samples and 4 were non-recurrent samples.
The disease-free recurrence period was 210 days, 318 days, 566 days, 761 days, and 1377 days for recurrent specimens, and 1594 days, 1599 days, 1835 days, and 2024 days for non-recurrent specimens, respectively.

<Preparation of measurement sample>
Nine specimens were solubilized as follows and used as measurement samples.

  Each specimen was cut to about 4 mm square or about 3 mm square on a petri dish placed on dry ice, and transferred to a microtube.

Next, a solubilizing reagent (50 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8.0), 50 mM NaF, 1 mM Na ₃ VO ₄ , 0.1% NP40, 0.2% protease inhibitor cocktail (sigma)) ) Was dispensed at 400 μL for 4 mm square specimens and 300 μL for 3 mm square specimens. Next, the sample tube was placed in a homogenizer and then homogenized, and centrifuged at 15000 rpm for 5 minutes at 4 ° C. with a micro high-speed centrifuge CF15RX (manufactured by Hitachi). Next, the supernatant of the sample tube was transferred to a 1.5 mL microtube placed on ice and used as a measurement sample.

<Measurement of GSTπ expression level>
Nine specimens for measurement prepared by the above method were measured by the method described in Example 1.

  The results of this example are shown in FIG. FIG. 6 shows a box whisker chart.

  The results shown in FIG. 6 indicate that among 9 samples, recurrent samples tend to have low GSTπ expression levels and non-recurrent samples tend to have high GSTπ expression levels. As a result, it was suggested that this tendency corresponds to that the GSTπ high expression strain in the result of Example 1 is highly sensitive to an anthracycline anticancer agent.

  (Example 3)

<Determination of Sensitivity of Cancer Cells to Anthracycline Anticancer Agent Based on GSTπ Expression Level and CDK2 Specific Activity Value / CDK1 Specific Activity Value>
Next, the determination of the sensitivity of an anthracycline anticancer agent in cancer cells was examined by combining the GSTπ expression level of cancer cells contained in a biological sample and the value of CDK2 specific activity value / CDK1 specific activity value.

<Measurement of IC50>
Each of the 29 breast cancer cell lines shown in Reference Example 1 was measured by the method described in Example 1. Here, the median value of IC50, 392 nM, was set as a threshold, a breast cancer cell line showing a threshold value or more was set as an insensitive strain, and a breast cancer cell line showing a threshold value less than the threshold was set as a sensitive strain.

<Measurement of GSTπ expression level>
Each of the 29 breast cancer cell lines shown in Reference Example 1 was measured by the method described in Example 1. Here, the threshold value for the expression level of GSTπ was set to 3, a breast cancer cell line exhibiting a threshold value or higher was designated as a GSTπ high expression strain, and a breast cancer cell line exhibiting less than the threshold was designated as a GST low expression strain.

<Measurement of CDK1 and CDK2 activity values>
The reagent to be used was prepared as follows.

(1) Membrane cleaning solution
10x TBS solution 100mL
900mL ultrapure water
(2) Immunoprecipitation buffer
1M Tris-HCl (pH7.4) 10.0mL
10% NP-40 Alternative PROTEIN GRADE (CALBIOCHEM) 2.0mL
10% sodium azide solution 1.9mL
Ultrapure water QS-200mL
(3) CDK antibody dilution
Sucrose (Kishida Chemical Co., Ltd.) 17.1g
1M Tris-HCl (pH7.4) 1.25mL
3M NaCl solution 2.5mL
10% sodium azide solution 470μL
Ultrapure water QS-50mL
(4) Anti-CDK1 antibody reagent
CDK antibody dilution 1.9mL
Anti-CDK1 antibody (448μg / mL) 17.5mL
190% of 10% sodium azide solution
Immunoprecipitation buffer 840 μL
(5) Anti-CDK2 antibody reagent
CDK antibody diluent 1.6mL
Anti-CDK2 antibody (360μg / mL) 8.3mL
10% sodium azide solution 100μL
Immunoprecipitation buffer 960μL
(6) Background antibody reagent
CDK antibody dilution 9.5mL
Rabbit IgG (CALBIOCHEM) (12.5mg / mL) 440μL
Immunoprecipitation buffer 960μL
(7) Immunoprecipitation washing solution 1
1M Tris-HCl (pH7.4) 25.0mL
10% NP-40 Alternative PROTEIN GRADE (CALBIOCHEM) 50.0mL
4.7% 10% sodium azide solution
Ultra pure water QS-500mL
(8) Immunoprecipitation washing solution 2
1M Tris-HCl (pH7.4) 12.5mL
3M NaCl solution 25.0mL
10% sodium azide solution 2.2mL
Ultrapure water QS-250mL
(9) Immunoprecipitation washing solution 3
1M Tris-HCl (pH7.4) 12.5mL
10% sodium azide solution 2.3mL
Ultrapure water QS-250mL
(10) Kinase reaction reagent ultrapure water 17.9mL
1M Tris-HCl (pH7.4) 1.22mL
10% sodium azide solution 200μL
1M Magnesium chloride solution 450μL
25 mM ATP-γS solution 1.8 mL
5 mg / mL Histone H1 solution 900 μL
(11) Fluorescence labeling reaction buffer
2.2M MOPS-NaOH (pH7.4) 34.1mL
0.5M EDTA (pH8.0) 2.5mL
2.1% 10% sodium azide solution
Ultrapure water QS-250mL
(12) Fluorescent labeling reagent
5- (Iodoacetamido) fluorescein (Molecular Probes) 25mg
DMSO 6mL
116mL fluorescent labeling reaction buffer
(13) Fluorescence labeling reaction stop solution
2.2M MOPS-NaOH (pH7.4) 455mL
N-Acetyl-L-Cysteine (Nacalai Tesque) 2.45g
450% of 10% sodium azide solution
Ultra pure water 43.5mL
(14) Fluorescence enhancement reagent
Blocking One (Nacalai Tesque) 100mL
10% sodium azide solution 10mL
Ultra pure water 890mL

  The activity values of CDK1 and CDK2 were measured as follows.

  Dispense 30 μL / well of 20% Beads Sol. (300 μL of protein A beads, 900 μL of immunoprecipitation buffer) to a filter plate (Millipore), and add anti-CDK1 antibody reagent, anti-CDK2 antibody reagent, and background antibody reagent. 90 μL was dispensed into each corresponding well. Next, each measurement sample of 29 types of breast cancer cell lines shown in Reference Example 1 prepared as in Reference Example 2 was dispensed at 30 μL / well, and 4 ° C. at PlateShaker (SHK-10, Masuda Rika Kogyo). After stirring for 2 hours, CDK1 and anti-CDK1 antibody, and CDK2 and anti-CDK2 antibody were reacted.

  After the reaction, washing was performed as follows. After the reagent in each well was aspirated, 200 μL / well of immunoprecipitation washing solution 1 was dispensed and aspirated. Next, 200 μL / well of immunoprecipitation washing solution 2 was dispensed and aspirated. Next, 200 μL / well of immunoprecipitation washing solution 3 was dispensed and aspirated.

After washing, 50 μL / well of Kinase reaction reagent was dispensed, and the mixture was stirred with Plate Shaker (SI-300C, manufactured by AS ONE) at 37 ° C., 900 rpm for 1 hour. Remove the filter plate from the plate shaker, set the collection plate (Bibby Sterilin) under the filter plate, and centrifuge at 4 ° C, 2000 rpm for 5 minutes in a centrifuge (BECKMAN COURTER Allegra ^TM 6KR Centrifuge). did.

  After centrifugation, the activity measurement reaction product collected on the above-described collection plate was further transferred to a reaction plate (Applied Biosystem) at 14 μL / well, and a fluorescent labeling reagent was dispensed at 14 μL / well. After dispensing, the mixture was shielded from light and stirred with a plate shaker at 25 ° C. and 400 rpm for 20 minutes.

  After stirring, 200 μL / well of a fluorescent labeling reaction stop solution was dispensed onto the reaction plate, and the solution in each well of the reaction plate was further transferred to a measurement filter plate (Millipore) at 100 μL / well. Next, the measurement plate was washed twice by sucking the whole solution in each well and then dispensing and aspirating the membrane washing solution at 200 μL / well. After washing, the fluorescence enhancing reagent was dispensed 200 μL / well and aspirated five times and dried.

  After drying, a plate reader (Infinite F200, manufactured by Tecan) was used to perform fluorescence analysis of the measurement plate, and the activity value of CDK1 and the activity value of CDK2 in each measurement sample were calculated based on the calibration curve. In addition, the calibration curve was prepared by performing fluorescence analysis by the above method using a solution containing each protein (Recombinant CDK1 / cyclin B1, active or Rccombinant CDK2 / cyclin E, active) at three concentrations. . The measured CDK1 activity and CDK2 activity 1U (unit) are values indicating fluorescence intensity equivalent to the fluorescence amount when the protein is 1 ng.

  <Measurement of CDK1 and CDK2 expression levels>

  The reagent to be used was prepared as follows.

(1) Blocking reagent
BSA 10.0g
10x TBS solution 25mL
212.8g of ultrapure water
10% sodium azide aqueous solution 2.25mL
(2) Membrane cleaning solution
10x TBS Solution 100mL
900mL ultrapure water
(3) Anti-CDK1 antibody reagent
Block Ace (Dainippon Sumitomo Pharma Co., Ltd.) 2.4g
72.3g of ultrapure water
10% sodium azide aqueous solution 750μL
Anti-CDK1 antibody (0.455mg / mL) (final concentration 12μg / mL) 1.98mL
(4) Anti-CDK2 antibody reagent
Block Ace (Dainippon Sumitomo Pharma Co., Ltd.) 2.4g
72.4g of ultrapure water
10% sodium azide aqueous solution 750μL
Anti-CDK2 antibody (0.306 mg / mL) (final concentration 7.5 μg / mL) 1.94 mL
(5) Secondary antibody reagent
BSA 2.5g
10x TBS solution 25mL
216.3g of ultrapure water
10% aqueous solution of sodium azide (final concentration 0.1%) 2.21mL
Biotinylated anti-rabbit IgG (Southern Biotech) (500μg / mL, final concentration 8μg / mL) 4.00mL
(6) Fluorescent labeling reagent
BSA 2.5g
10x TBS solution 25mL
217.8g of ultrapure water
10% sodium azide aqueous solution (final concentration 0.1%) 2.23mL
Fluorescein-StreptAvidin (Vector) (1mg / mL, final concentration 10μg / mL) 2.50ml
(7) Fluorescence enhancement reagent
3-Morpholinopropanesulfonic acid (NW.209.25) (Dojindo Laboratories) 41.9g
Sodium azide 2.0g
6N sodium hydroxide solution 34g
Ultra pure water QS-2L
(8) Sample diluent for measurement (first time)
Membrane cleaning solution 900μL
10% NP-40 Alternative PROTEIN GRADE (CALBIOCHEM) 100μL
(9) Sample diluent for measurement (second time)
Membrane cleaning solution 19.9mL
10% NP-40 Alternative PROTEIN GRADE (CALBIOCHEM) 100μL

  The expression levels of CDK1 and CDK2 were measured as follows.

  The measurement samples of 29 types of breast cancer cell lines shown in Reference Example 1 prepared according to Reference Example 2 were diluted to a membrane washing solution of 475 μL, a measurement sample diluent of 300 μL, and a measurement sample of 25 μL. Next, 100 μL / well of the diluted measurement sample was dispensed onto the filter plate and aspirated.

  After aspiration, 300 μL / well of the membrane washing solution was dispensed onto the filter plate and again aspirated for washing.

  Next, the blocking reagent was dispensed at 100 μL / well on the filter plate, and then blocked by aspiration.

  50 μL / well of CDK1 antibody reagent and CDK2 antibody reagent are respectively dispensed and aspirated into wells corresponding to CDK1 and CDK2 on the filter plate, and then again each 50 μL / well of CDK1 antibody reagent and CDK2 antibody reagent are dispensed. The reaction was allowed to stand at 23 ° C. for 2 hours on Shaker (SI-300C, manufactured by Tecan). Next, the reagent in the well was aspirated, 300 μL / well of the membrane washing solution was dispensed, and aspirated and washed. Washing was performed 4 times.

  After washing, the wells corresponding to CDK1 and CDK2 on the filter plate are treated with the secondary antibody reagent in the same manner as the above dispensing of the CDK1 antibody reagent and the CDK2 antibody reagent, and the primary antibody and the secondary antibody are reacted. And washed. The standing time here was 45 minutes, and washing was performed twice.

  A fluorescent labeling reagent was dispensed at 100 μL / well onto a filter plate and aspirated. Further, 300 μL / well of the membrane cleaning solution was dispensed and aspirated for cleaning. Washing was performed 4 times. Next, the fluorescent enhancement reagent was dispensed at 200 μ / well onto the filter plate, and after suction, it was dried.

  After drying, fluorescence analysis of the measurement plate was performed using Plate Reader (Infinite F200, manufactured by Tecan), and the expression level of CDK1 and the expression level of CDK2 in each measurement sample were calculated based on the calibration curve. In addition, the calibration curve is for each of the four concentrations (10% NP-40 Alternative PROTEIN GRADE (CALBIOCHEM), CDK1 / CyclinB1, active (UPSTATE), or cdk2 (1-298) (SantaCruz Solution)) was prepared by performing fluorescence analysis by the method described above.

  <Calculation of CDK1 and CDK2 specific activity values>

  The specific activity values of CDK1 and CDK2 were measured as follows. From the activity value of CDK1, the expression level of CDK1, the activity value of CDK2 and the expression level of CDK2 measured by the above method, the following formula:

CDK1 specific activity value = CDK1 activity value / CDK1 expression level
CDK2 specific activity value = CDK2 activity value / CDK2 expression level

  The CDK1 specific activity value (mU / ng) and the CDK2 specific activity value (mU / ng) were determined by the above calculation, and the CDK2 specific activity value / CDK1 specific activity value was calculated.

  Here, a threshold value of CDK2 specific activity value / CDK1 specific activity value is 7.41, a breast cancer cell line showing a threshold value or higher is defined as a CDK2 specific activity value / CDK1 specific activity value high value strain, and a breast cancer cell showing less than the threshold value The strain was defined as a CDK2 specific activity value / CDK1 specific activity value low value strain.

  In this example, among the samples for measurement of each of the 29 breast cancer cell lines shown in Reference Example 1, the specific activity values of CDK1 and CDK2 could not be calculated for the two cell lines MDA-MB-415 and UACC812. It was. Thus, the results for 27 breast cancer cell lines excluding the two cell lines are shown in FIG.

  From the results shown in FIG. 7, among the 27 types of breast cancer cell lines, the GSTπ high expression strain or the CDK2 specific activity value / CDK1 specific activity high value strain tended to be highly sensitive to doxorubicin. As a result, when the GSTπ expression level of the cancer cell is large or when the CDK2 specific activity value / CDK1 specific activity value of the cancer cell is larger than the threshold value, it can be determined that the cancer cell is highly sensitive to the anthracycline anticancer agent. It was done. Moreover, the GSTπ low expression strain and the CDK2 specific activity value / CDK1 specific activity low value strain tended to be less sensitive to doxorubicin. As a result, it was shown that when the expression level of GSTπ in the cancer cells is small and the CDK2 specific activity value / CDK1 specific activity value of the cancer cells is smaller than the threshold, it can be determined that the sensitivity to the anthracycline anticancer agent is low.

  (Example 4)

<Examination of combined effects of GSTπ expression level and CDK2 specific activity value / CDK1 specific activity value>
Next, based on the data obtained in Example 3, the effect of determination combining the expression level of GSTπ and the CDK2 specific activity value / CDK1 specific activity value was determined based on the determination result based on the expression level of GSTπ alone, CDK2 specific activity. The determination was performed using the determination result based on the combination of the value / CDK1 specific activity value alone, the combination of the expression level of GSTπ and the CDK2 specific activity value / CDK1 specific activity value.

  <Examination of GSTπ expression level alone>

  Table 2 shows the GSTπ expression level and IC50 results for each of the 27 breast cancer cell lines in Example 3. Here, the threshold value of the GSTπ expression level was 8.05, a breast cancer cell line showing a threshold value or higher was regarded as a sensitive strain, and a breast cancer cell line showing less than the threshold was regarded as an insensitive strain.

  The odds ratio in the results shown in Table 2 was 4.8, P by logistic regression was 0.15, and AUC by ROC analysis was 0.604.

  Table 3 shows the CDK2 specific activity value / CDK1 specific activity value and IC50 results for each of the 27 breast cancer cell lines in Example 3. Here, the threshold value of the CDK2 specific activity value / CDK1 specific activity value was set to 13.0, a breast cancer cell line showing a threshold value or higher was regarded as a sensitive strain, and a breast cancer cell line showing a threshold value less than the threshold was designated as an insensitive strain.

  The odds ratio in the results shown in Table 3 was 12.0, P by logistic regression was 0.0117, and AUC by ROC analysis was 0.712.

  <Examination by combination of GSTπ expression level and CDK2 specific activity value / CDK1 specific activity value>

  Table 4 shows the combinations of the GSTπ expression level and the CDK2 specific activity value / CDK1 specific activity value for the 27 breast cancer cell lines in Example 3 and the IC50 results. Here, a breast cancer cell line having a threshold value of GSTπ expression level of 8.05 or higher or a CDK2 specific activity value / CDK1 specific activity value threshold value of 13.0 or higher is defined as a sensitive strain, and the GSTπ expression level is less than 8.05 and CDK2 specific activity value / CDK1. A breast cancer cell line showing a specific activity value less than 13.0 was defined as an insensitive strain.

  The odds ratio in the results shown in Table 4 was 30.0, P by logistic regression was 0.0004, and AUC by ROC analysis was 0.819.

  When comparing the odds ratios corresponding to the risk ratios from Table 2 to Table 4 and the results obtained, the values of GSTπ expression level and CDK2 specific activity value / CDK1 specific activity value are higher than the predicted odds ratios of the individual. The odds ratio combined with was higher than the combined odds ratio expected alone. From these results, it was suggested that the ability to judge the sensitivity of cancer cells to anthracycline synergistically improves by combining the expression level of GSTπ and the value of CDK2 specific activity value / CDK1 specific activity value.

Claims (8)

Measuring the expression level of GSTπ contained in a biological sample containing cancer cells of breast cancer;
The expression level of GSTπ obtained in the measurement step is compared with the first threshold value. When the expression level of GSTπ is equal to or higher than the first threshold value , the cancer cell of the breast cancer contained in the biological sample is added to the anthracycline anticancer agent. Determining that the sensitivity is high;
A method for determining the sensitivity of cancer cells of breast cancer contained in a biological sample to an anthracycline anticancer agent. The method according to claim 1, wherein the anthracycline anticancer agent is daunorubicin, doxorubicin, pirarubicin, aclarubicin, epirubicin, oxaunomycin or idarubicin. The method according to claim 1 or 2 , wherein the anthracycline anticancer agent is doxorubicin. The measurement step further measures the specific activity value of CDK1 and the specific activity value of CDK2 contained in the biological sample containing cancer cells of breast cancer,
Determining step compares the value with the second threshold value of specific activity value of CDK1 obtained by CDK2 specific activity value / measurement process obtained in measurement step (CDK2 specific activity value / CDK1 specific activity value), When the value of CDK2 specific activity value / CDK1 specific activity value is not less than the second threshold value , it is determined that the sensitivity of the cancer cells of breast cancer contained in the biological sample to an anthracycline anticancer agent is high . The method according to any one of the above. In the determination step, when the expression level of GSTπ obtained in the measurement step is less than the first threshold value and the value of the CDK2 specific activity value / CDK1 specific activity value is less than the second threshold value , The method according to claim 4, wherein it is determined that the sensitivity of the cancer cell to the anthracycline anticancer agent is low. Computer
An acquisition means for acquiring an expression level of GSTπ contained in a biological sample containing cancer cells of breast cancer;
A comparison means for comparing the expression level of GSTπ with the first threshold;
When the expression level of GSTπ is equal to or higher than the first threshold value, a determination unit that determines that a breast cancer cell contained in a biological sample is highly sensitive to an anthracycline anticancer agent;
An output means for outputting the determination result;
Computer program to function as. The computer further calculates the value of CDK2 specific activity value / CDK1 specific activity value (CDK2 specific activity value / CDK1 specific activity value) from the CDK1 activity value, CDK1 expression level, CDK2 activity value and CDK2 expression level. Function as a calculation means for calculating
The obtaining means further obtains the activity value of CDK1, the expression level of CDK1, the activity value of CDK2 and the expression level of CDK2 in a biological sample containing cancer cells of breast cancer,
The comparison means further compares the CDK2 specific activity value / CDK1 specific activity value with the second threshold value,
Determining means, when the value of C DK2 specific activity value / CDK1 specific activity value is equal to or higher than the second threshold value, it determines that the susceptibility to breast cancer cells anthracycline anticancer agent is highly contained in the biological sample,
The computer program according to claim 6 . Determining means further expression level is less than the first threshold GSTpi, and when the value of the CDK2 specific activity value / CDK1 specific activity value is less than the second threshold value, breast cancer contained in the biological sample of the cancer cells It is determined that the sensitivity to anthracycline anticancer agents is low.
The computer program according to claim 7 .

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